WUSCHEL-RELATED HOMEOBOX 2 is important for protoderm and suspensor development in the gymnosperm Norway spruce

Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive.

R E S E A R C H A R T I C L E Open Access

WUSCHEL-RELATED HOMEOBOX 2 is

important for protoderm and suspensor

development in the gymnosperm Norway

spruce

Tianqing Zhu*, Panagiotis N Moschou, José M Alvarez, Joel J Sohlberg and Sara von Arnold

Abstract

Background: Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are

involved in patterning and morphogenesis of the early embryo in Arabidopsis However, the role of WOX genes in other taxa, including gymnosperms, remains elusive Here, we use somatic embryos and reverse genetics for

studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce)

Results: The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was

reduced Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation

Conclusions: Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants In addition, PaWOX2 might exert a unique function in suspensor

expansion in gymnosperms

Keywords: Norway spruce, Protoderm, Somatic embryo, WUSCHEL-RELATED HOMEOBOX 2

Background

The basic plant body pattern is set up during

embryo-genesis In seed plants, this body plan has been

de-scribed as the superimposition of two patterns: an

apical-basal and a radial pattern [1] The molecular

pro-cesses that establish this primary body plan have mainly

been studied in the angiosperm model species Arabidopsis

(Arabidopsis thaliana) In contrast, knowledge about

the molecular regulation of embryo development in

gymnosperms is limited, partly owing to the lack of identified zygotic embryo defective mutants and gen-etic tractability However, by using somatic embryos and reverse genetics it has been possible to study the regulation of embryo development in conifers [2] We are studying the early stages of embryo development

in Norway spruce and especially the role of members

of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, which encode transcription factors that play important roles in the determination of cell fate during embryogenesis in angiosperms [3, 4]

Angiosperms and gymnosperms separated approxi-mately 300 million years ago and expectedly their

* Correspondence: Tianqing.zhu@slu.se

Department of Plant Biology, Uppsala BioCenter, Swedish University of

Agricultural Sciences and Linnean Center for Plant Biology, PO-Box 7080,

SE-75007 Uppsala, Sweden

© 2016 Zhu et al Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

patterning during embryogenesis differs significantly For

convenience, embryogenesis in Arabidopsis can be

di-vided into three general phases, described as

proembryo-geny, early embryogeny (globular-stage to heart-stage

transition) and late embryogeny [5] Proembryogeny

be-gins after fertilization An asymmetric cell division of the

zygote generates a smaller apical cell and a larger basal

cell The apical cell is the founder of the embryo proper,

while the basal cell develops into the suspensor The

ap-ical cell undergoes several rounds of stereotyped

asym-metric divisions, giving rise to cells with positionally

determined cell fate At the beginning of early

embry-ogeny, in the 8-cell embryo proper, a single round of

tangential divisions separate the outer layer of eight cells

from the eight inner cells [6] The inner cells are founder

cells of the ground tissue and vascular elements The

outer cells form the protoderm which will become the

epidermis Plant epidermis is characterized by the

secre-tion of lipids and waxes to its outer cell wall [7] The

continuous hydrophobic layer forms the cuticle Owing

to the periclinal cell division pattern in the protoderm,

the protodermal cells remain essentially separated from

the inner cells throughout embryogenesis Root and

shoot meristems are established during the transition

from globular to heart-stage After the heart-stage the

suspensor is degraded by programmed cell death [5]

During late embryogeny, there is a switch from pattern

formation to storage product accumulation

In conifers, the sequence of embryo development can

also be described by three phases: proembryogeny, early

embryogeny, and late embryogeny [8] Proembryogeny

begins when the zygote undergoes several rounds of

nuclear duplications The produced free nuclei are first

arranged in a tier before cellularization After a cell

div-ision, two tiers are formed The cells in the upper tier

elongate to form a functional suspensor and the cells in

the lower tier divide creating the embryonal mass

(analogous to the embryo proper in angiosperms) Early

embryogeny begins with the elongation of the embryonal

suspensor Cells in the outer layer of the embryonal

mass divide mainly anticlinally, but also periclinally

giv-ing rise to additional internal layers [8], unlike

angio-sperms where only anticlinal cell divisions take place

Nevertheless, the outer cell layer in the embryonal mass

in conifer embryos defines a functional protoderm [9, 10]

Late embryogeny is a period of histogenesis and

organo-genesis Early during this phase, the suspensor cells are

dismantled by programmed cell death [11], and the root

and shoot apical meristems are delineated

Members of the WOX gene family play important

roles in determining cell fate during plant development

Phylogenetic analyses have identified three major clades

in the WOX gene family: the modern (WUS and

WOX1-7), the intermediate (WOX8, 9, 11, and 12) and the

ancient clade (WOX10, 13, and 14) [12] In the gymno-sperm Norway spruce (Picea abies), 11 WOX genes that belong to all major clades, have been identified [13]

All WOX genes examined show very specific expres-sion patterns, both spatially and temporally, which are important for their molecular functions [14] WOX2, WOX8 and WOX9 have been implicated in patterning and morphogenesis of the early embryo in Arabidopsis [3, 15, 16] AtWOX2 is expressed in the egg cell and the zygote [3] After the first cell division, AtWOX2 marks the apical cell, while, AtWOX8 and AtWOX9 mark the basal cell AtWOX8 and AtWOX9 share redundant func-tions [16] The Arabidopsis wox8wox9 double mutant shows aberrant orientation of cell division planes in the embryo [16] Previously, we showed that the Norway spruce gene PaWOX8/9 is the orthologue of AtWOX8 and AtWOX9 [17] Similarly to Arabidopsis, down-regu-lation of PaWOX8/9 causes disturbed orientation of the cell division plane and cell fate determination during early embryo pattern formation suggesting that PaWOX8/9 ex-erts an evolutionarily conserved function

It has previously been shown that PaWOX2, which is highly similar in sequence to AtWOX2, is expressed dur-ing embryo development in gymnosperms [13, 18–20] Herein we report that PaWOX2 is transiently expressed

in the embryonal mass and in the upper part of the suspensor in early and early late embryos Furthermore,

by using reverse genetics, we functionally characterize PaWOX2 Down-regulation of PaWOX2 during develop-ment of early embryos results in aberrant early embryos, which lack distinct embryonal mass and suspensor do-mains, a proper protoderm, and have shorter suspensor cells This suggests an evolutionarily conserved function

of WOX2 in protoderm formation early during embryo development among seed plants In addition, PaWOX2 might exert a unique function in proper suspensor cell expansion in gymnosperms

Materials and methods

Plant material

The embryogenic cell line 61:21 of Norway spruce (Picea abiesL Karst) has been used in this study The cell line was established as described by Högberg et al [21] The cell line was stored in liquid nitrogen and thawed a couple of months before the start of experiments After thawing the cell cultures were maintained as described previously [2] Briefly, proembryogenic masses (PEMs) were maintained on solidified proliferation medium con-taining the plant growth regulators (PGRs) auxin and cytokinin To stimulate development of somatic embryos the cultures were first transferred to pre-maturation medium lacking PGRs for one week and then to matur-ation medium containing 30 μM abscisic acid (ABA)

Early embryos (EEs) differentiated after one week on

maturation medium; early late embryos (LE1s) and LE2s

developed after two and three weeks on maturation

medium, respectively; maturing embryos (ME1s),

charac-terized by the initiation of cotyledons, developed after

five weeks on maturation medium ME2s (almost fully

matured embryos) and ME3s (fully matured embryos)

de-veloped after about eight weeks on maturation medium

RNA extraction, cDNA synthesis and quantitative

real-time PCR

Samples for analyzing the mRNA level of PaWOX2

(accession number: AM286747) during embryo

develop-ment, were collected from nine sequential

developmen-tal stages: PEM1 (after seven days on proliferation

medium), PEM2 and PEM3 (after three and seven days

on pre-maturation medium respectively), EE, LE1, LE2,

ME1, ME2 and ME3 (after one to eight weeks on

matur-ation medium) The sampling was performed at mid-day

and samples were frozen in liquid nitrogen and stored

at −80 °C after collection

Total RNA was isolated using the Spectrum Plant

Total RNA kit (Sigma-Aldrich, USA) according to the

manufacturer’s instructions For each sample, 1 μg of

total RNA was reverse transcribed with RevertAid H

Minus First Strand cDNA Synthesis Kit (Fermentas,

Thermo Scientific, Sweden) using an equimolar ratio of

random and oligo-dT primers according to the

manufac-turer’s instructions

Quantitative real-time PCR (qRT-PCR) was performed

as described previously [17] Three reference genes,

(PaPHOS) were used [22] Two to three biological

repli-cates, each with three technical replicates were

per-formed for each test The primer sequences are

presented in Additional file 1: Table S1 Statistical

ana-lysis was done by t-test

RNA in situ hybridization

For RNA in situ hybridization (ISH) the following

mate-rials were used: ovules from cones collected in the end

of June and somatic embryos (EEs, LE1s and ME1s) The

ovules were fixed and embedded as described by Karlgren,

et al [23] The somatic embryos were fixed in 3.7 %

for-maldehyde, 5.0 % acetic acid and 50 % ethanol overnight

and embedded in Technovit 8100 A gene-specific

frag-ment was used as a probe The probes were prepared with

DIG RNA Labeling Kit (see primer sequences in

Add-itional file 1: Table S1) (Sigma-Aldrich, USA) In situ

hybridization was performed essentially as described by

Karlgren et al [23] Sections of 10μm were hybridized to

digoxigenin-labeled RNA probes The pictures were

proc-essed using Adobe Photoshop CS6 13.0 software

RNA interference vector construction

The coding sequence (CDS) of PaWOX2 was amplified from a cDNA library of early somatic embryos of Norway spruce [13] The full-length CDS was subcloned into the pJET1.2/blunt cloning vector using the CloneJET™ PCR Cloning Kit (Fermentas, Thermo Scientific, Sweden) To obtain RNA interference (RNAi) constructs, two overlap-ping fragments of PaWOX2 were amplified and fused to form a hairpin structure for PaWOX2 (Additional file 2: Figure S1) To fuse these fragments, EcoRI and BamHI di-gestion sites were added on forward primers as linkers The hairpin was confirmed by sequencing Primers are presented in Additional file 3: Table S2

Hairpin structures were introduced into pENTR™/D-TOPO® (Invitrogen, Carlsbad, CA, USA) and then inserted by att site LR recombination into the destin-ation vector pMDC7 [LexA-VP16-ER (XVE) β-estradiol inducible promoter, which is derived from the pER8 vector and contains the estrogen receptor-based transac-tivator XVE] [24, 25] or pMDC32 (35S constitutive promoter) [26] Hairpin structures were confirmed by sequencing Vectors were introduced by electroporation into Agrobacterium tumefaciens strain GV3101

Transgenic cell lines

Embryogenic cultures were transformed by co-cultivation with A tumefaciens as described previously [17] Stably transformed lines were selected after four weeks Genomic DNA was isolated from PEMs from selected lines by using the DNeasy plant mini kit (Qiagen, Germany), according

to the manufacturer’s instructions Transformed lines were confirmed by PCR

The mRNA level of PaWOX2 in PaWOX2 RNAi lines was analyzed by qRT-PCR In the case of the inducible XVE-WOX2i lines, cultures were induced with β-es-tradiol (10 μM) for 48 h before the analysis The lines 35S:WOX2i.2, 35S:WOX2i.3, 35S:WOX2i.4 and XVE-WOX2i.12 were selected for further studies (Additional file 4: Figure S2) The untransformed 61:21 line was used

as a control In addition, in the time-laps tracking ex-periments we also included a transformed control line (T-control), expressing the reporter GUS (β-glucuron-idase) under the 35S promoter

To study if PaWOX2 regulates cell division, the

[PaRETINOBL ASTOMA-RELATED PROTEIN-LIKE (PaRBRL), PaEXTRA SPINDLE POLES (PaESP), two E2F family genes (PaE2FABL) and five CYCLIN-LIKE (PaCYCLs) genes] were analysed in EEs from the control and line 35S:WOX2i.4 by qRT-PCR as previously described [17]

Morphological analysis

Samples of EEs for morphological analysis were collected from the control and lines 35S:WOX2i.2, 35S:WOX2i.3,

35S:WOX2i.4 and XVE-WOX2i.12 after one week on

maturation medium The samples were embedded by

mixing with 2 ml of 1.2 % (w/v) Seaplaque agarose

(FMC BioProducts, USA) in 60 mm Petri dishes The

length and width of suspensor cells of about 40 EEs

from the control and 35S:WOX2i lines were measured

using the ImageJ software (ver 1.48 g) [27]

To further study embryo morphology, 26 LE1s from the

control and line 35S:WOX2i.4 were scanned with a Zeiss 780

confocal microscope (Carl Zeiss AG), using the 488 nm

Argon laser line, and the 20x objective (NA = 0.80)

For histological analysis, EEs from the control and line

35S:WOX2i.4 were fixed in 3.7 % formaldehyde, 5.0 %

acetic acid and 50 % ethanol overnight Subsequently,

samples were dehydrated in 50, 75, 90 and 100 %

etha-nol series Finally, the samples were embedded in

Tech-novit 8100 (Kulzer, Wehrheim, Germany) The embryos

were processed for serial sectioning (10 μM) on a Zeiss

HM 355 microtome

The cuticle of untreated LE1s from the control and

line 35S:WOX2i.4 was stained in freshly prepared Oil

Red, 0.2 % (w/v) in water, for 5 min and then washed in

water [28] The stained embryos were hand-sectioned

and examined under a Zeiss Axioplan microscope in

dark field with a 5x objective (NA = 0.12)

Time-lapse tracking analysis was performed to

exam-ine in great detail the developmental pattern from EE to

ME EEs from the controls (both untransformed and

T-control) and lines 35S:WOX2i.2, 35S:WOX2i.3 and

35S:WOX2i.4 (50 embryos per line) were sampled

after one week on maturation medium and

trans-ferred to fresh maturation medium Embryo

morph-ology was examined every second day for 15 days

To study the effect of PaWOX2 on the maturation

process, after one week on pre-maturation medium

35S:WOX2i.3, 35S:WOX2i.4 and XVE-WOX2i.12 were

re-suspended in liquid pre-maturation medium and

plated out as a thin layer on filter paper placed on

mat-uration medium For the XVE-WOX2i line, β-estradiol

(10 μM) was added to the maturation medium, either

from the start or after two weeks on maturation

medium, when LE2s had already developed The

devel-opment of embryos was recorded for 14 days on

matur-ation medium The time points examined were 1, 3, 6,

10 and 14 days The number of ME3s developed per

ini-tial gram of tissue was estimated after seven weeks on

maturation medium

Results

Expression of PaWOX2 during embryo development

It has been shown that PaWOX2 is specifically expressed

in early somatic embryos of Norway spruce [13, 19] In

order to get a higher resolution of the fluctuations of the

mRNA level of PaWOX2 during embryo development, samples from nine sequential developmental stages, spanning all three phases of embryo development, were collected for qRT-PCR analysis (Fig 1a) The mRNA level of PaWOX2 was low in PEMs and increased sharply upon formation of EEs (Fig 1b) The highest mRNA level of PaWOX2 was observed in LE1s (Fig 1b) Thereafter, it decreased, to become almost undetectable

in MEs (Fig 1b)

To gain more insight into the spatial expression of PaWOX2 in situmRNA hybridization was conducted on somatic embryos PaWOX2 mRNA was detected in the embryonal mass and in the upper part of the suspensor

in both EEs and LE1s (Fig 2) No signal could be detected in MEs (data not shown) In situ mRNA localization analyses were also conducted on ovules collected in the end of June at the time when zygotic

Hybridization signals were detected in late embryos (Additional file 5: Figure S3A), but not in maturing embryos (Additional file 5: Figure S3C) In addition, signals were detected in the mega gametophyte resid-ing in the front of the growresid-ing embryo (Additional file 5: Figure S3)

PaWOX2 is required for proper protoderm formation

In order to examine the function of PaWOX2, we con-structed stably transformed PaWOX2 RNAi lines, using constitutive (35S; referred as 35S:WOX2i) or inducible (XVE; referred as XVE-WOX2i) promoters to drive ex-pression of a hairpin RNA used to promote PaWOX2 specific RNAi The down-regulation of PaWOX2 was confirmed by qRT-PCR (Additional file 4: Figure S2) The transcript level of PaWOX2 in non-induced tissue was lower than in the control In accordance, it was pre-viously shown that the XVE promoter is partially active

in Norway spruce even in the absence of β-estradiol [18]

A typical Norway spruce EE has a polarized structure with a distinct border between the compact globular em-bryonal mass in the apical part and elongated suspensor cells in the basal part (Fig 3a.1) The embryonal mass consists of densely cytoplasmic cells delineated by a dis-tinct protoderm with a smooth surface (Fig 3a.3) Ap-proximately 80 % of EEs from the control and 50 % of EEs from the 35S:WOX2i lines had normal morphology (Fig 3b, Additional file 6: Table S3) The aberrant EEs in the control were characterized by a successive transition from small meristematic cells in the embryonal mass to elongated cells in the suspensor (Fig 3a.5) The aberrant EEs in the 35S:WOX2i lines failed to establish distinct embryonal mass and suspensor domains In these em-bryos the border between the embryonal mass and the suspensor was severely disturbed (Fig 3a.4 and 6) In

about one-third of the EEs in 35S:WOX2i lines the

em-bryonal mass lacked a smooth surface and vacuolated

cells were formed both from the outer cell layer and

within the embryonal mass (Fig 3a.2 and 6, Additional

file 7: Table S4) This phenotype was rarely found in the

control Furthermore, the length of the suspensor cells

in EEs of 35S:WOX2i lines were significantly reduced

(Fig 3c) Normal and aberrant LE1s were examined by

confocal laser scanning microscopy (Fig 4) The

proto-derm covering the embryonal mass on LE1s from the

control was arranged in a structured way (Fig 4)

Proto-dermal cells were characterized by a rectangular

cell-shape In contrast, the embryonal mass in aberrant LE1s

from the 35S:WOX2i lines lacked a distinct protoderm

and a smooth surface The aberrant morphology could

also be observed in LE2s (data not shown)

We used Oil Red staining to detect presence of the cuticularized layer in LE1s [28] The staining was strong

on surface of the embryonal mass in LE1s from the con-trol, but weak and patchy on surface of LE1s from the 35S:WOX2i line (Fig 5) This staining pattern indicates that the cuticularized layer is poorly developed around the embryonal mass in LE1s from the 35S:WOX2i line

PaWOX2 is not regulating cell division at the transcriptional level

Previously, we showed that PaWOX8/9, which specifies the cell division plane orientation in the basal part of the embryonal mass, regulates cell division at the transcrip-tional level [17] In order to elucidate whether PaWOX2 exerts a similar function, we compared the mRNA abun-dance of nine cell-cycle-regulating genes in EEs from the

Fig 1 Relative mRNA level of PaWOX2 a Schematic representation of the developmental stages of embryo development Proliferating proembryogenic masses (PEMs): PEM1, seven days after subculture to fresh proliferation medium in the presence of plant growth

regulators (PGRs), PEM2 and PEM3, three and seven days after transfer to pre-maturation medium lacking PGRs; early embryo (EE), after one week on maturation medium; early late embryo (LE1) and late embryo before the formation of cotyledons (LE2) after one and two weeks

on maturation medium, respectively; maturing embryo (ME1), characterized by the initiation of cotyledons after five weeks on maturation medium; ME2 (almost fully maturated embryo) and ME3 (fully maturated embryo) after about eight weeks on maturation medium The majority

of the embryos in each sample represented the designated specific developmental stage However, since the development was not strictly synchronized, embryos from previous and subsequent stages could also be represented b qRT-PCR analysis of the relative mRNA level of PaWOX2 in nine sequential developmental stages of embryo development The mRNA level is relative to the mRNA level of PaWOX2 in LE1 and

is normalized against three reference genes: CELL DIVISION CONTROL 2 (PaCDC2), ELONGATION FACTOR-1 (PaEF1) and PHOSPHOGLUCOMUTASE (PaPHOS) The mRNA level is the mean ± SE of three biological replicates Different letters indicate significant differences in the mRNA levels among developmental stages (ANOVA, p < 0.05)

control and from line 35S:WOX2i.4 by qRT-PCR No

significant differences in mRNA level of the tested

cell-cycle-regulating genes could be observed between

con-trol and the RNAi line (Additional file 8: Figure S4) The

previous suggests that PaWOX2 and PaWOX8/9

regu-late the expression of different targets during embryo

development

PaWOX2 is important for the development of mature

embryos

To gain information about how the aberrations observed

during early embryogenesis in PaWOX2 RNAi lines

affect further development of these embryos, we

per-formed time-lapse tracking analysis during maturation

Individual EEs were selected from the control and

PaWOX2 RNAi lines Three developmental pathways

were observed: i) normal embryo maturation (Fig 6a,

Normal), ii) embryo degeneration, in which the cells on

the surface layer of the embryonal mass became

vacu-olated, and new embryogenic tissue was initiated from

the degenerated embryos (Fig 6a, Degeneration), and

iii) development of ball-shaped embryos, in which the

embryos lacked differentiated cotyledons (Fig 6a,

Ball-shaped) Most of the EEs from the control developed

into normal mature embryos (76 %), and only 4 %

followed the degeneration pathway (Fig 6b, Additional

file 9: Table S5) About 50 % of the EEs from

35S:WOX2i lines developed normally The frequency

of EEs which developed into ball-shaped embryos was

significantly higher in the 35S:WOX2i lines than in the

control However, an increased frequency of ball-shaped

embryos was also observed in the transformed control (Additional file 9: Table S5), indicating that the formation

of ball-shaped embryos is an artifact of the transgenic se-lection process An increase in the frequency of ball-shaped embryos in transgenic lines of Norway spruce has been reported before [29] Interestingly, the frequency of EEs that degenerated (on average 20 %) was significantly higher in the 35S:WOX2i lines, suggesting that this path-way is caused by down-regulation of PaWOX2

In order to examine the importance of PaWOX2 for

35:WOX2i lines were plated out as a thin layer on maturation medium Control cultures ceased to prolif-erate during the first three days EEs and LE1s were formed after six days and developed into MEs within two weeks (Fig 7a) In contrast, in 35S:WOX2i lines, the proliferation of the embryogenic tissue did not decline on maturation medium Although some LE1s had developed after six days (Fig 7a), only a few LE1s developed into MEs Consequently, the yield of MEs was significantly lower in 35S:WOX2i lines than

in the control (Fig 7b, Additional file 10: Table S6) However, the MEs formed had a normal morphology and 76 % germinated as compared to 80 % of MEs in the control line (data not shown)

For elucidating when during embryo development PaWOX2 is important, we analyzed development of embryos of the inducible XVE-WOX2i lines Initially

12 XVE-WOX2i lines treated with β-estradiol were screened for their potential to develop MEs In 8 out of these 12 lines, the cultures continued to proliferate on

Fig 2 Expression pattern of PaWOX2 in early (EE) and early late (LE1) embryos according to mRNA in situ localization Hybridization signals appear as dark blue in bright field microscopy a Longitudinal section of an EE b Longitudinal section of a LE1 Note that signals are detected in the embryonal mass and in the upper part of the suspensor in both EE and LE1 The insert presents the sense probe control No signal was detected after hybridization with sense probe (negative control) EM, embryonal mass; S, suspensor Bar = 50 μm

Fig 3 (See legend on next page.)

maturation medium and the yield of MEs was low Cell

line XVE-WOX2i.12 was choosen for further studies

The frequency of EEs with normal morphology was

significantly lower in line XVE-WOX2i.12 induced

with β-estradiol than in the control (Fig 3b) For

examining the role of PaWOX2 in the formation of

MEs, line XVE-WOX2i.12 was induced with

β-estra-diol either from the first or from the third week on

maturation medium When down-regulation of PaWOX2

was induced from the first week on maturation medium, the yield of MEs was significantly decreased (Fig 7c, Additional file 11: Table S7) However, the yield of MEs was not affected when down-regulation of

matur-ation medium, when LE2s had formed These results suggest that the transient high expression of PaWOX2 during the formation of EEs and LE1s is important for further development of the embryos

(See figure on previous page.)

Fig 3 PaWOX2 is important for the development of normal early embryos (EEs) EEs were sampled from the control and lines XVE-WOX2i.12, 35S:WOX2i.2, 35S:WOX2i.3 and35S:WOX2i.4 after one week on maturation medium a A.1, Normal EE from the control Note the smooth surface of the embryonal mass and the distinct border between the embryonal mass and the suspensor A.2, Aberrant EE from line 35S:WOX2i.4 Note the vacuolated cells in the outer cell layer of the embryonal mass and the lack of a distinct border between the embryonal mass and the suspensor A.3, longitudinal section of a normal EE from the control A5, longitudinal section of an aberrant EE from the control A.4 and A.6, longitudinal sections of aberrant EEs from line 35S:WOX2i.4 Note the vacuolated cells in the embryonal mass and the lack of distinct protoderm, owing to aberrant cell division of the cells in the outer cell layer of the embryonal mass denoted by arrows ⇧ Bar, 50 μm b Frequency of normal EEs in the control and lines XVE-WOX2i.12, 35S:WOX2i.2, 35S:WOX2i.3 and35S:WOX2i.4 [non-induced (−), induced (+) with β-estradiol] More than 100 EEs were analyzed from each line (Additional file 6: Table S3) Data are presented as means ± SE of three biological replicates Asterisks indicate significant differences in the frequency of normal embryos between the control ( −) and the transgenic lines (one tail t-test, p < 0.05) c Boxplot presenting the average length of about 50 suspensor cells from EEs in the control and line 35S:WOX2i.4 Box plot shows the median (solid line), the 25th and 75th percentiles (boxes), and the 5th and 95th percentiles (error bars) The presented data are based on about 20 embryos Asterisk indicates significant difference in the length of suspensor cells between the control and the transgenic line (ANOVA, p < 0.05)

Fig 4 PaWOX2 is important for correct differentiation of the protoderm Images of LE1s in the control and line 35S:WOX2i.4 were acquired by laser scanning confocal microscopy Cells are visible due to their intrinsic autofluorescence, when excited with strong intensity argon laser line at

488 nm Images are pseudocolored green Upper tier, maximum projection of a Z-stack (30 sequential stack images, 2 μm each optical section); Bottom tier, single optical layer images of the embryos shown in the upper tier Bar, 50 μm

In this study we show that the mRNA level of PaWOX2

in somatic embryos is transiently up-regulated during

early and late embryogeny Results obtained by

qRT-PCR analyses, were in good agreement with results

ob-tained by in situ mRNA hybridization The expression of

PaWOX2 in EEs and LE1s was detected in the

embry-onal mass and in suspensor cells that are in proximity to

the embryonal mass In situ mRNA analyses were also

conducted on zygotic embryos Owing to the difficulty

to obtain high quality sections of the zygotic embryos

we could not localize the signal to specific cell-types However, we could detect signal in late embryos but not

in maturing embryos A comparable expression pattern for PaWOX2 in somatic and zygotic embryos of Norway spruce has been reported by Palovaara, et al [19] Taken together, these results indicate that PaWOX2 is expressed in a similar way in somatic and zygotic em-bryos during embryo development

Hybridization signals could be detected in the region

of the mega gametophyte that resides in front of the growing embryo In Scots pine (Pinus sylvestris) it has

Fig 5 PaWOX2 is important for the formation of a cuticularized layer Freshly isolated LE1s from the control (1, 3 and 5) and line 35S:WOX2i.4 (2, 4 and 6) were stained with Oil Red 1 and 2, intact embryos; 3 and 4, longitudinal hand-sectioned embryos; 5 and 6, transverse hand-sectioned embryos Note the strong positive staining on the surface of LE1s from the control, denoted by arrowheads Bar, 50 μm

Fig 6 Developmental pathways of early embryos (EEs) in the control and line 35S:WOX2i.4 a The developmental pathway of EEs was followed by time-lapse tracking analysis during 15 days EEs from the control cultures and line 35S:WOX2i.4 were isolated after one week on maturation medium and transferred to fresh maturation medium Representative images are shown for days 1, 3, 5, 7, 9, 11 and 15 The embryos followed three developmental pathways: i) normal development; ii) degeneration, in which the cells on the surface layer of the embryonal mass became vacuolated after five days, followed by initiation of new embryogenic tissue after nine days (the areas in the white squares are presented in higher magnification in the inserts; ⇧ arrows denote vacuolated surface layer); iii) development of ball-shaped embryos The ball-shaped

embryos did not develop further after 9 days Bar, 100 μm b Frequency of EEs that developed normally (black bars), went through the degeneration pathway (grey bars) or developed into ball-shaped embryos (white bars) The presented frequencies are based on the development of 80 –90 embryos (Additional file 9: Table S5) Data are presented as means ± SE of three biological replicates Asterisks indicate significant differences in the frequency of embryos in each developmental pathway between the control and the transgenic line (one tail t-test, p < 0.05)